ERbB-4 targeted ribozymes

ABSTRACT

Enzymatic RNA molecules which cleave ErbB-4 mRNA and uses thereof.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to international application No.PCT/US98/23279 filed Oct. 30, 1998, which in turn claims priority toprovisional application No. 60/063,875 filed Oct. 31, 1997.

INTRODUCTION

This invention relates to methods for inhibition of growth oftransformed cells, and treatment and diagnosis of diseases andconditions related to ErbB-4 expression.

The epidermal growth factor (EGF) receptors have been implicated inhuman cancer more frequently than any other family of growth factorreceptors. The EGF receptor gene is often amplified or overexpressed insquamous cell carcinoma and glioblastomas [Jenkins et al. (1989) CancerGenet. Cytogenet. 39:253]. Similarly, ErbB-4 is overexpressed inadenocarcinomas of the stomach, breast and ovary.

The epidermal growth factor receptor (EGFR/ErbB) family is a group oftyrosine kinases that is frequently overexpressed in a variety ofcarcinomas [Gullick, W. J. (1991) Br. Med. Bull. 47:87-98; Hynes, N. E.and Stern, D. F. (1994) Biochem. Biophys. Acta 1198:165-184; Lemoine, N.R. et al. (1992) Br. J. Cancer 66:1116-1121]. This class I subfamily ofreceptors is comprised of four members: EGFR [Xu, Y. H. et al. (1984)Nature 309:806-810], HER2/ErbB-2/neu [Schechter, A. L. et al. (1984)Nature 312:513-516], HER3/ErbB-3 [Kraus, M. H. et al. Proc. Natl. Acad.Sci. USA 86:9193-9197; Plowman, G. D. et al. (1990) Proc. Natl. Acad.Sci. USA 87:4905-4909], and HER4/ErbB-4 [Plowman, G. D. et al. (1993)Proc. Natl. Acad. Sci. USA 90:1746-1750]. Data from numerouslaboratories suggest that the EGFR family members may play a complexrole in signaling [Wada, T. et al. (1990) Cell 61:1339-1347; Goldman, R.et al. (1990) Biochemistry 29:11024-11028; Caraway, K. L. and Cantley L.C. (1994) Cell 78:5-8]. Most human breast cancer cells express more thanone of the EGF family receptors, and different combinations of receptorscan heterodimerize or homodimerize. These receptor interactions lead tothe activation of multiple signaling pathways and contribute to thepathogenicity and tumorigenicity of breast cancer [Earp, S. H. et al.(1995) Breast Cancer Resarch and Treatment].

A number of growth factors, classified as EGF-like ligands, have beenidentified that bind and stimulate the kinase activity of EGF-familyreceptors. EGF, transforming growth factor α (TGFα), amphiregulin (AR),heparin-binding EGF(HB-EGF), and betacellulin (BTC) have been describedas specific for EGFR [Savage, C. R. et al. (1972) J. Biol. Chem.241:7612-7621; Marquardt, H. et al. (1983) Science 223:1079-1082;Shoyab, M. et al. (1989) Science 243:1079-1082; Higashiyama, S. et al.(1991) Science 251: 936-939; shing, Y. et al. (1993) Science259:1604-1607]. Several differentially spliced variants, named heregulin(HRG) also known as neuregulin (NRG), or neu differentiation factor(NDF) [Holmes, W. E. et al. (1992) Science 256:1205-1210; Wen, D. et al.(1992) Cell 69:559-572], acetylcholine-receptor inducing activity (ARIA)[Falls, D. G. et al. (1993) Cell 72:801-815], glial growth factor (GGF)[Marchionni, M. A. et al. (1993) Nature (London)362: 312-318] and gp30[Lupu, R. et al. (1990) Science 249:1552-1555], were initiallyidentified as candidate neu ligands by their ability to induce neutyrosine phosphorylation [Peles, E. and Yarden, Y. (1993) Bioassays15:815-824]. However, recent results demonstrate that ErbB-3 and ErbB-4are primary receptors for heregulin [Plowman, G. D. et al. (1993) Nature366:473-475; Carraway, K. L. III et al. (1994) J. Biol. Chem. 269:14303-14306]. Activation of ErbB-2 by HRG is thought to occur throughtransphosphorylation resulting from heterodimerization with eitherErbB-3 or ErbB-4 [Tzahar, E. et al. (1994) J. Biol. Chem.269:40:25226-25223; Peles, E. et al. (1993) EMBO J. 12:961-971;Sliwkowski, M. X. et al. (1994) J. Biol. Chem. 269: 14661-14665]. Mostrecently, betacellulin was also shown to activate the ErbB-4 receptor ina Ba/F3 system [Riesell, D. J. et al. (1996) Oncogene 12: 245-353].

Amplification and/or overexpression of EGFR and ErbB-2 are clearlyimportant factors in neoplastic transformation of breast epithelium[Jardines, L. et al. (1993) Pathobiology 61:268-282]. Elevated ErbB-4levels have been found in certain breast cancer cell lines [Plowman, G.D. et al. (1993) Proc. Natl. Acad. Sci. USA 90:1746-1750], but little isknown about the expression or the clinical significance of ErbB-4receptors in the diagnosis and prognosis of human breast cancer.

SUMMARY OF THE INVENTION

To investigate the biological significance of ErbB-4 in human breastcancer, we used molecular targeting of the ErbB-4 mRNA by ribozymes. Wedescribe the generation of three ribozymes (Rz6, Rz21, Rz29) targeted tospecific sites within the ErbB-4 mRNA open reading frame. We demonstratethat all three ErbB-4 ribozymes cleave ErbB-4 mRNA precisely andefficiently under physiological conditions in this cell free system. Wealso illustrate the intracellular efficacy and specificity of the ErbB-4ribozymes in a model system (32D cell system). 32D cells are a murinehematopoietic IL3-dependent cell line that does not express detectablelevels of endogenous EGF-family receptors. Overexpression of ErbB-4receptors in 32D cells (32D/ErbB-4) abrogated IL-3-dependence bystimulation with NRG. We show that two of the ErbB-4 ribozymes (Rz6 andRz29) were able to down-regulate ErbB-4 expression and were capable ofabolishing the neuregulin-induced mitogenic effect in 32D/ErbB-4 cells.These results demonstrate that ribozyme Rz29 and Rz6 are biologicallyfunctional ribozymes.

Therefore, this invention relates to ribozymes, or enzymatic RNAmolecules, directed to cleave mRNA species encoding specific sites inErbB-4. In particular, applicants describe the selection and function ofribozymes capable of cleaving this RNA and their use to reduce activityof ErbB-4 in various tissues to treat the diseases discussed herein,more particularly, breast cancer. Such ribozymes are also useful fordiagnostic applications.

Ribozymes are RNA molecules having an enzymatic activity which is ableto repeatedly cleave other separate RNA molecules in a nucleotide basesequence specific manner. Such enzymatic RNA molecules can be targetedto virtually any RNA transcript and efficient cleavage has been achievedin vitro [Jefferies, et al. (1989) Nucleic Acid Res. 17:1371].

Ribozymes act by first binding to a target RNA. Such binding occursthrough the target RNA binding portion of a ribozyme which is held inclose proximity to an enzymatic protion of the RNA which acts to cleavethe target RNA. Thus, the ribozyme first recognizes and then binds atarget RNA through complementary base-pairing, and once bound to thecorrect site, acts enzymatically to cut the target RNA. Strategiccleavage of such a target RNA will destroy its ability to directsynthesis of an encoded protein. After a ribozyme has bound and cleavedits RNA target it is released from that RNA to search for another targetand can repeatedly bind and cleave new targets.

The enzymatic nature of a ribozyme is advantageous over othertechnologies, such as antisense technology (where a nucleic acidmolecule simply binds to a nucleic acid target to block its translation)since the effective concentration of ribozyme necessary to effect atherapeutic treatment is lower than that of an antisenseoligonucleotide. This advantage reflects the ability of the ribozyme toact enzymatically. Thus, a single ribozyme molecule is able to cleavemany molecules of target RNA. In addition, the ribozyme is a highlyspecific inhibitor, with the specificity of inhibition depending notonly on the base pairing mechanism of binding to the target RNA, butalso on the mechanism of target RNA cleavage. Single mismatches, orbase-substitutions, near the site of cleavage can completely eliminatecatalytic activity of a ribozyme. Similar mismatches in antisensemolecules do not prevent their action [Woolf, T. M. et al. (1992) Proc.Natl. Acad. Sci. USA 89:7305-7309]. Thus, the specificity of action of aribozyme is greater than that of an antisense oligonucleotide bindingthe same RNA site. Consequently, the ribozyme agent will only affectcells expressing that particular gene, and will not be toxic to normaltissues.

The invention can be used to treat cancer or pre-neoplastic conditions.Two preferred administration protocols can be used, either in vivoadministration to reduce the tumor burden, or ex vivo administration toeradicate transformed cells from tissues such as bone marrow prior toimplantation.

Thus, in the first aspect the invention features an enzymatic RNAmolecule (or ribozyme) which cleaves mRNA associated with development ormaintenance of cancer, e.g. those mRNAs produced from the gene ErbB4including mRNA targets disclosed in Table 1.

TABLE 1 Nucleotide mRNA target sequence ID NOs  (60) GAUUUGGGUCUGGUGAGSEQ ID NO:1 (210) UGAGGUUGUCAUGGGC SEQ ID NO:2 (290) GUCACAGGCUACGUGUUAGSEQ ID NO:3

Hammerhead ribozymes (Rz) targeted to sites within ErbB-4 mRNA describedin Table 1 were generated. These ErbB-4 ribozymes (Rz6, Rz21, Rz29)effectively catalyzed the precise cleavage of ErbB-4 mRNA underphysiological conditions in a cell-free system. One of these ribozymes,Rz29, down-regulated ErbB-4 receptor expression by as much as 65%, witha corresponding 10-fold decrease in ErbB-4 tyrosine phosphorylation in a32D cell model system. Furthermore, expression of this functional ErbB-4ribozyme in T47D and MCF-7 human breast carcinoma cells led to adown-regulation of endogenous of ErbB-4 expression and a reduction ofanchorage-independent colony formation.

By “enzymatic RNA molecule” it is meant an RNA molecule which hascomplementarity in a substrate binding region to a specified mRNAtarget, and also has an anzymatic activity which is active tospecifically cleave that mRNA. That is, the enzymatic RNA molecule isable to intermolecularly cleave mRNA and thereby inactivate a targetmRNA molecule. This complementarity functions to allow sufficienthybridization of the enzymatic RNA molecule to the target RNA to allowthe cleavage to occur. One hundered percent complementarity ispreferred, but complementarity as low as 50-75% may also be useful inthis invention.

Ribozymes that cleave the specified sites in ErbB4 RNAs represent anovel therapeutic approach for the treatment of tumors and otherconditions where overexpression of ErbB-4 is causal such as childhoodmedulloblastoma [Gilbertson, R. J. et al. (1998) Cancer Res.58:3932-3941]. Applicants show that ribozymes are able to inhibit theactivity of ErbB4 and that the catalytic acitiviy of the ribozymes isrequired for their inhibitory effect. Those of ordinary skill in theart, will find that it is clear from the examples described that otherribozyems that cleave these sites in ErbB4 RNAs may be readily designedand are within the scope of this invention.

In a second aspect, the invention features a mammalian cell whichincludes an enzymatic RNA molecule as described above. Preferably, themammalian cell is a human cell.

In a third aspect, the invention features an expression vector whichincludes nucleic acid encoding an enzymatic RNA molecule describedabove, located in the vector, e.g., in a manner which allows expressionof that enzymatic RNA molecule within a mammalian cell.

In a fourth aspect, the invention features a method for treatment ofbreast cancer by administering to a patient an enzymatic RNA molecule asdescribed above.

The enzymatic RNA molecules of this invention can be used to treat humanbreast cancer. Such treatment can also be extended to other relatedgenes in nonhuman primates. Affected animals can be treated at the timeof cancer detection or in a prophylactic manner. This timing oftreatment will reduce the number of affected cells.

BRIEF DESCRIPTION OF THE DRAWINGS

These and other features, aspects, and advantages of the presentinvention will become better understood with reference to the followingdescription, appended claims, and accompanying drawings where:

FIG. 1. Illustrates the ErbB-4 ribozymes targeted sequences and cleavagesites (seq ID No. 1,2,3) down-stream of the translation initiation sitein the ErbB-4 mRNA open reading frame. ECD, extracellular domain, TM,transmembrane domain, CYD, cytoplasmic domain.

FIGS. 2, A and B. Catalytic activity of ErbB-4 ribozyme in anextracellular system. Lane 1 represents molecular weight markers. Lane2, ³²P-labeled ErbB-4 transcript with an expected size of 622nucleotides (nts). Lanes 3-5, cleavage products of the three ErbB-4ribozymes (Rz6: 518, 110nts; Rz21: 285, 337nts; Rz29: 232, 390nts).Lanes 6, 7, mutant ribozymes do not cleave ErbB-4 transript. B) Lane 1,molecular weight marker. Lane 2, ³²P-labeled ErbB-3 transcript with anexpected size of 698 nucleotides.

FIG. 3. Growth assay: 32D cells were plated at a density of 1×10⁴cells/ml in IL3 free medium, medium supplemented with IL-3, or in mediumlacking IL-3 but supplemented with 100 ng/ml of human recombinant HRG.Viable cells were counted on day 3 after seeding. Heregulin can induceIl-3-independent growth in 32D/E4 and 32D/E2+E3 cells. All samples wereprepared in triplicate. This assay was repeated more than three times.The SD was within 10%.

FIG. 4. Regulation of receptor tyrosine phosphorylation by HRG in 32D/E4and 32D/E2+E3 cells. 500 ug of lysates from untreated or HRG (100 ng/mlfor 5 minutes) treated 32D transfectants (32D/wt, 32D/E2, 32D/E3,32D/E4, 32D/E2+E3) were immunoprecipitated with anti-receptor antibodies(αE2, αE3, αE4). 32D/EGFR cells (E1) were treated with 100 ng/ml of EGFfor 5 minutes and immunoprecipitated with anti-EGFR antibody (αE1).Lysates from EGF or HRG-treated cells are denoted ‘+’ while lysates fromuntreated cells are denoted ‘−’. The precipitates were then subjected toWestern blotting with an anti-phosphotyrosine antibody (UBI, LakePlacid, N.Y.). MW, Molecular Weight; IP, immunoprecipitation.

FIG. 5. ErbB-4 ribozyme abolishes HRG-induced mitogenesis.32D-transfected cells were plated at a density of 1×10⁴ cells with orwithout IL-3, or with 100 ng/ml HRG in the absence of IL-3. Two daysafter plating, the cells were labeled with ³[H]thymidine for two hours.³[H] thymidine incorporation was then analyzed by scintillationcounting. The parental 32D cells are labeled as ‘wt’. 32D/E4 transfectedcells are denoted as E4. E4+V represents the empty vector transfected32D/E4 cells. Ribozyme transfected cells are indicated as Rz6, Rz21 andRz29. Rz29 abolished the HRG induced IL-3-independent growth. Allsamples were prepared in triplicate. This assay was repeated threetimes. The SD was within 10%.

FIGS. 6A-D. Rz29 down-regulation of ErbB-4 expression in 32D/ErbB-4cells. The levels of ErbB-4 in 32D/E4 and Rz29 transfected 32D/E4 cellswere quantitatively measured by flow-cytometry. 1×10⁶ cells wereharvested and stained with an anti-ErbB-4 monoclonal antibody incombination with fluorescence-labeled anti-mouse IgG antibody andanalyzed by FACScan. A) Expression of ErbB-4 in vector-transfected cells(E4/V). Right-hand curves, specific staining; left-hand curves,nonspecific staining (primary antibody omitted); ordinates, relativecell number; abscissas, log fluorescence. B) Rz29 down-regulates ErbB-4expression by 50%. Dotted-line curve, ErbB-4 expression in ErbB-4/Vcells. Solid-line curve, ErbB-4 expression in Rz29-transfected cells. C)Rz 21 has no effect on ErbB-4 expression. Dotted-line curve, ErbB-4expression in ErbB-4/V cells. Solid-line curve, ErbB-4 expression inRz21 transfected cells. D) Rz6 down-regulates ErbB-4 expression by 30%.Dotted-line curve, ErbB-4 expression in ErbB-4/V cells. Solid-linecurve, ErbB-4 expression in Rz6 transfected cells.

FIG. 7. Reduction of autophosphorylation of ErbB-4 receptor by Rz29ribozyme. Cells were treated with (+) or without (−) HRG (100 ng/ml) for5 minutes prior to lysis, and 400 ug of lysates were immunoprecipitatedwith specific anti-ErbB-4 antibody. Precipitated proteins were then usedfor in vitro kinase assay as described in Material and Methods below.Lysates from 32D wild-type and transfected cells are indicated above.32D, untransfected cells; E4, ErbB-4 transfected cells; Rz29, ErbB-4ribozyme Rz29-expressing 32D/E4 cells; V, cells transfected with emptyvector. Molecular weight standards are shown on the left-hand side ofthe gel.

FIG. 8 ErbB-4 ribozyme down-regulation of endogenous ErbB-4 expressionin T47D human breast cancer cells. The level of ErbB-4 in T47D/wt andT47D/Rz pool clones were quantitatively measured by flow-cytometry.1×10⁶ cells were harvested and stained with an anti-ErbB-4 monoclonalantibody in combination with fluorescence-labeled anti-mouse IgGantibody and analyzed by FACScan. Left-hand curve (thin line curve)represents nonspecific staining (primary antibody omitted). Right-handcurve (Bold line curve) represents the ErbB-4 expression in T47Dwild-type cells. The dotted-line curve (middle curve) represents theErbB-4 expression in Rz6 transfected cells. The ordinates, relative cellnumber; abscissas, log fluorescence.

FIG. 9. Anchorage-independent growth assay: Expression of ErbB-4ribozyme in T47D cells (T47D/Rz6 pool clone) inhibits colony formationby more than 50%. A bottom layer of 0.1 ml Iscove's modified Eagle'smedium (IMEM) containing 0.6% agar and 10% FCS was prepared in 35 mmtissue culture dishes. After the bottom layer solidified, cells (10,000per dish) were added on a 0.8 ml top layer containing 0.4% Bacto Agar,and 5% FCS. All samples were prepared in triplicate. The cells wereincubated for approximately 12 days at 37° C. Colonies larger than 60 umwere counted in a cell colony counter.

FIG. 10. Reduction of NRG and BTC induced ErbB-4 autophosphorylation inT47D/Rz transfected cells. Cells were treated with or without NRG1-αandBTC (100 ng/ml) for 5 minutes prior to lysis, and 400 ug of lysates wereimmunoprecipitated with a specific anti-ErbB-4 antibody. Precipitatedproteins were then subjected to Western blotting with ananti-phosphotyrosine antibody (UBI). Lane 1: Molecular weight standards.Lane 2,5,8 are untreated samples. Lane 3,6,9 are the lysates fromT47D/wt, T47D/Rz cells treated with 100 ng/ml of NRG1-α. Lane 4, 7, 10are the lysates from T47D/wt, T47D/vector and T47D/Rz cells treated with100 ng/ml of BTC. Down-regulation of ErbB-4 in T47D cells dramaticallyreduced NRG and BTC induced ErbB-4.

FIG. 11. Growth effects of ErbB-4 ribozyme on T47D cells.Anchorage-independent growth assays: Expression of the ErbB-4 ribozymein T47D cells inhibits colony formation, independent of colony size. Abottom layer of 0.1 ml IMEM containing 0.6% agar and 10% FCS wasprepared in 35 mm tisssue culture dishes. After the bottom layersolidified, cells (10,000 per dish) were than added in a 0.8 ml toplayer, containing 0.4% Bacto Agar, and 5% FCS. All samples were preparedin triplicate. The cells were incubated for approximately 12 days at 37°C. Colonies larger than 60 um, 80 um, 100 um, and 120 um were counted bya cell colony counter.

FIG. 12. Growth effects of ErbB-4 ribozyme on MCF-7 cells.Anchorage-independent growth assays: Expression of the ErbB-4 ribozymein MCF-7 cells inhibits colony formation, independent of colony size. Abottom layer of 0.1 ml IMEM containing 0.6% agar and 10% FCS wasprepared in 35 mm tissue culture dishes. After the bottom layersolidified, cells (10,000 per dish) were than added in a 0.8 ml topelayer, containing 0.4% Bacto Agar, and 5% FCS. All samples were preparedin triplicate. The cells were incubated for approximately 12 days at 37°C. Colonies larger than 120 um, 140 um, and 160 um were counted by acell colony counter.

FIG. 13. Down-regulation of endogenous ErbB-4 expression in T47D cellsstrongly inhibited NRG-induced colony formation. Anchorage-independentgrowth assays: A bottom layer of 0.1 ml IMEM containing 0.6% agar and10% FCS was prepared in 35 mm tissue culture dishes. After the bottomlayer solidified, cells (10,000 per dish) were than added in a 0.8 mltop layer, containing 0.4% Bacto Agar, and 5% FCS and 100 ng/ml ofEGF-like ligands. All samples were prepared in triplicate. The cellswere incubated for approximately 15 days at 37° C. Colonies larger than60 um were counted by a cell colony counter. The blank bars representthe T47D wild type cells. The open bars represent the ErbB-4 ribozymetransfected T47D cells. In the wild type cells, NRG had most effect on acolony formation amount the EGF-like ligands. In ribozyme transfectedcells, BTC had the dominant effect and NRG-stimulated colony formationwas reduced by 70%.

FIG. 14. Growth of T47Dwt and two ErbB-4 ribozyme transfectants inathymic nude mice.

FIG. 15. ErbB-4-ribozyme mediated down-regulation of ErbB-4 in MCF-7cells resulted in reduction of tumor growth in vivo. 5×10⁶ MCF-7 wildtype cells, as well as the ribozyme transfected cells were implanted inovariectomized mice. With estradiol treatments, the MCF-7 wild typecells, as well as the empty vector transfected cells grew large tumorsto a mean tumor size of 2000±200 mm³ (filled and open circles). Incontrast, tumor growth of ribozyme expressing MCF-7 cells wassignificantly inhibited (p<0.001; student's test) with a mean tumor sizeof 600±74 mm³(triangles and squares).

DETAILED DESCRIPTION OF THE INVENTION

In a preferred embodiment the invention provides a method for producinga class of enzymatic cleaving agents which exhibit a high degree ofspecificity for the RNA of a desired target. The enzymatic nucleic acidmolecule is preferably targeted to a highly conserved sequence region oftarget mRNAs encoding ErbB4 proteins such that specific treatment of adisease or condition can be provided with either one or severalenzymatic nucleic acids. Such enzymatic nucleic acid molecules can bedelivered exogenously to specific cells as required. Alternatively, theribozymes can be expressed from DNA/RNA vectors that are delivered tospecific cells.

In one of the preferred embodiments of the invention, the enzymaticnucleic acid molecule is formed in a hammerhead or hairpin motif, butmay also be formed in a motif of a hepatitis d virus, group I intron,group II intron or RNaseP RNA (in association with an RNA guidesequence) or Neurospora VS RNA. Examples of such hammerhead motifs aredescribed by Rossi et al. (1992) AIDS Research and Human Retroviruses8:183; of haripin motifs by Hampel and Tritz, (1989) Biochemistry28:4929, Feldstein et al. (1989) Gene 82:53; Haseloff and Gerlach (1989)Gene 82:43, and Hampel et al. (1990) Nucleic Acids Res. 18:299; of thehepatitis d virus motif is described by Perrotta and Been (1992)Biochemistry 31:16; of the RNaseP motif by Guerrier-Takada et al. (1983)Cell 35:849, Forster and Altman (1990) Science 249:783, Li and Altman(1996) Nucleic Acids Res. 24:835; Neurospora VS RNA ribozyem motif isdescribed by Collins (Saville and Collins, (1990) Cell 61:685-696;Saville and Collins, (1991) Proc. Natl. Acad. Sci. USA 88:8826-8830;Collins and Olive, (1993) EMBO J. 14:363); Group II introns aredescribed by Griffin et al. (1995) Chem. Biol. 2:761, Michels and Pyle(1995) Biochemistry 34:2965; and the Group I introns by Cech et al. U.S.Pat. No. 4,987,071. These specific motifs are not limiting in theinvention and those skilled in the art will recognize that all that isimportant in an enzymatic nucleic acid molecule of this invention isthat it has a specific substrate binding site which is complementary toone or more of the target gene RNA regions, and that it have nucleotidesequences within or surrounding that substrate binding site which impartan RNA cleaving activity to the molecule.

Synthesis of nucleic acids greater than 100 nucleotides in length isdifficult using automated methods, and the therapeutic cost of suchmolecules is prohibitive. In this invention, small nucleic acid motifs(e.g. antisense oligonucleotides, hammerhead or the hairpin ribozymes)are used for exogenous delivery. The simple structure of these moleculesincreases the ability of the nucleic acid to invade targeted regions ofthe mRNA structure. However, these nucleic acid molecules can also beexpressed within cells from eukaryotic promoters [Kashani-sabet et al.(1992) Antisense Res. Dev. 2:3-15; Dropulic et al. (1992) J. Virol.66:1432-1441; Weerasinghe et al. (1991) J. Virol. 65:5531-5534; Ojwanget al. (1992) Proc. Natl. Acad. Sci. USA 89:10802-10806; Chen et al.(1992) Nucleic Acids Res. 20:4581-4589; Thompson et al. (1995) NucleicAcids Res. 23:2259]. Those skilled in the art realize that any nucleicacid can be expressed in eukaryotic cells from the appropriate DNA/RNAvector. The activity of such nucleic acids can be augmented by theirrelease from the primary transcript by a ribozyme [Ohkawa et al. (1992)Nucleic Acids Symp. Ser. 27:15-16; Taira et al. (1991) Nucleic AcidsRes. 19:3249-3255; Chowrira et al. (1994) J. Biol. Chem. 269:25856].

Such ribozymes are useful for the prevention of the diseases andconditions discussed above, and any other diseases or conditions thatare associated with the levels of ErbB4 activity in a cell or tissue. By“associated” is meant that the inhibition of ErbB4 RNAs and thusreduction in the level of respective protein activity will relieve tosome extent the symptoms of the disease or condition. It may also meanthat the occurence of such symptoms is correlated with the level of suchRNAs.

Target Sites

Ribozymes targeting selected regions of mRNA associated with tumor cellgrowth are chosen to cleave the target RNA in a manner which preferablyinhibits translation of the RNA. Genes are selected such that ihibitionof translation will preferably inhibit cell replication, e.g. byinhibiting producition of a necessary protein. Selection of effectivetarget sites within these critical regions of mRNA entails testing theaccessibility of the target RNA to hybridization with variousoligonucleotide probes. These studies can be performed using RNA probesand assaying accessibility by cleaving the hybrid molecule with RNAseH.Alternatively, such a study can use ribozyme probes designed fromsecondary cleavage products by polyacrylamide gel electrophoresis(PAGE), to detect the presence of cleaved and uncleaved molecules.

The following is but one example of a method by which suitable targetsites can be identified and is not limiting in this invention.Generally, the method involves identifying potential cleavage sites fora hammerhead ribozyme, and then testing each of these sites to determinetheir suitability as targets by ensuring that secondary structureformation is minimal.

The mRNA sequences are compared in an appropriate target region.Putative ribozyme cleavage sites are found. These sites represent thepreferable sites for hammerhead ribozyme cleavage within these twotarget mRNAs.

The sequence of human and mouse ErbB-4 mRNA can be screened foraccessible sites using a computer folding algorithm. Hammerhead orhairpin ribozyme cleavage sites are identified and are shown in Table 1.Other sites include all the GUX potential sites in the ErbB-4 mRNA.While mouse and human sequences can be screened and ribozyems thereafterdesigned, the human targeted sequences are of most utility. However,mouse targeted ribozymes are useful to test efficacy of action of theribozyme prior to testing in humans.

Hammerhead ribozymes are designed that could bind and are individuallyanalyzed by computer folding (Jaeger et al. (1989) Proc. Natl. Acad.Sci. USA 86:7706-7710) to assess whether the ribozyme sequences foldinto the appropriate secondary structure. Those ribozymes withunfavorable intramolecular interactions between the binding arms and thecatalytic core are eliminated from consideration. Varying binding armlengths can be chosen to optimize activity. Generally, at least 5 baseson each arm are able to bind to, or otherwise interact with, the targetRNA.

DNA oligonucleotides representing potential hammerhead or hairpinribozyme cleavage sites are synthesized. A polymerase chain reaction isused to generate a substrate for T7 RNA polymerase transcription fromhuman or murine ErbB-4 cDNA clones. Labeled RNA transcripts aresynthesized in vitro from the two templates. The oligonucleotides andthe labeled transcripts are annealed, RNAseH is added and the mixturesare incubated for the designated times at 37° C. Reactions are stoppedand RNA separated on sequencing polyacrylamide gels. The percentage ofthe substrate cleaved is determined by autoradiographic quantitationusing a Phosphor Imaging system. From these data, hammerhead or hairpinsites are chosen as the most accessible.

Ribozymes of the hammerhead or hairpin motif are desined to anneal tovarious sites in the mRNA message. The binding arms are complementary tothe target site sequences described above.

The ribozymes can be produced by gene transcription as described byCech, supra, or by chemical synthesis as described by Usman et al.(1987) J. Am. Chem. Soc. 109:7845-7854 and in Scaringe t al. (1990)Nucleic acids Res. 18:5433-5441 and U.S. Pat. No. 5,599,704 to Thompsonet al. and makes use of common nucleic acid protecting and couplinggroups such as dimethoxytrityl at the 5′-end, and phosphoramidites atthe 3′-end. The average stepwise coupling yields were >98%. Hairpinribozymes are synthesized in two parts and annealed to reconstruct theactive ribozyme [Chowrira and Burke (1992) Nucleic Acids Res.20:2835-2840]. Hairpin ribozymes are also synthesized from DNA templatesusing bacteriophage T7 RNA polymerase [Milligan and Uhlenbeck (1989)Methods Enzymol. 180:51]. All ribozymes are modified to enhancestability by modification of five ribonucleotides at both the 5′ and 3′ends with 2′-O-methyl groups. Ribozymes are purified by gelelectrophoresis using general methods or are purified by high pressureliquid chromatography (HPLC) or other liquid chromatography techniques,employing reverse phase columns and anion exchangers on silica andpolymeric supports. The purified ribozymes are resuspended in water.

The sequences of chemically synthesized ribozymes useful in this studyare shown in Table I. Those in the art will recognize that thesesequences are representative only of many more such sequences where theenzymatic portion of the ribozyme (all but the binding arms) is alteredto affect activity. For example, stem-loop II sequence of hammerheadribozymes can be altered (substitution, deletion, and/or insertion) tocontain any sequences provided a minimum of two base-paired stemstructure can form. Similarly, stem-loop VI and VII can be altered(substitution, deletion, and/or insertion) to contain any sequence,provided a minimum of two base paired stem structure can form. Thesequences listed in Table I can be formed of ribonucleotides or othernucleotides or non-nucleotides. Such ribozymes are equivalent to theribozymes described specifically in the Table.

Ribozyme activity can be optimized, including altering the length of theribozyme binding arms (stems I and III), or chemically synthesizingribozymes with modifications that prevent their degradation by serumribonucleases [Perrault et al. (1990) Nature 344:565; Pieken et al.(1991) Science 253:314; Usman and Cedergren (1992) Trends in Biochem.Sci. 17:334]. Various other chemical modifications can be made to thesugar moeities of enzymatic RNA molecules. Modifications which enhancetheir efficacy in cells, and removal of stem II bases to shorten RNAsynthesis times and reduce chemical requirements are described in U.S.Pat. No. 5,334,711. All documents cited herein supra and infra arehereby incorporated in their entirety by reference thereto.

Selected ribozymes can be administered prophylactically, or to patientshaving breast cancer, e.g. by exogenous delivery of the ribozyme to aninfected tissue by means of an appropriate delivery vehicle, e.g. aliposome, a controlled release vehicle, by use of iontophoresis,electroporation or ion paired molecules, or covalently attached adducts,or by incorporation into other vehicles, such as hydrogels,cyclodextrins, biodegradable nanocapsules, bioadhesive microspheres, andother pharmacologically approved methods of delivery. For someindications, ribozymes may be directly delivered ex vivo to cells ortissues with or without the aforementioned vehicles. Alternatively, theRNA/vehicle combination is locally delivered by direct injection or byuse of a catheter, infusion pump or stent. Other routes ofadministration include intramuscular, intravascular, subcutaneous orjoint injection, aerosol inhalation, oral (tablet or pill form),topical, systemic, ocular, intraperitoneal and/or intrathecal.Expression vectors for immunization with ribozymes and/or delivery ofribozymes are also suitable. The dosage will depend upon the diseaseindication and the route of administration but should be between 100-200mg/kg of body weight/day. The duration of treatment will extend throughthe course of the disease symptoms, possibly continuously. The number ofdoses will depend upon disease delivery vehicle and efficacy data fromclinical trials. A more detailed description of delivery methods isfound in U.S. Pat. No. 5,599,704 by Thompson et al.

Another means of accumulating high concentrations of a ribozyme withincells is to incorporate the ribozyme-encoding sequences into a DNAexpression vector. Transcription of the ribozyme sequences is drivenfrom a promoter for eukaryotic RNA polymerase I (pol I), RNA polymeraseII (pol II), or RNA polymerase III (poly III). Transcripts from pol IIor pol III promoters will be expressed at high levels in all cells; thelevels of a given pol II promoter in a given cell type will depend onthe nature of the gene regulatory sequences (enhancers, silencers, etc.)present nearby. Prokaryotic RNA polymerase promoters are also used,providing that the prokaryotic RNA polymerase enzyme is expressed in theappropriate cells [Gao and Huang (1993) Nucleic Acids Res. 21:2867-2872;Lieber et al. (1993) Methods Enzymol. 217:47-66]. Several investigatorshave demonstrated that ribozymes expressed from such promoters canfunction in mammalian cells [Lisziewicz et al. (1993) Proc. Natl. Acad.Sci. USA 90:8000-8004]. The above ribozyme transcription units can beincorporated into a variety of vectors for introduction into mammaliancells, including but not restricted to, plasmid DNA vectors, viral DNAvectors, (such as adenovirus or adeno-associated virus vectors), orviral RNA vectors (such as retroviral or alphavirus vectors). Viralvectors have been used to transfer genes and lead to either transient orlong term gene expression [Zabner et al. (1993) Cell 75:207; Carter(1992) Curr. Opi. Biotech. 3:533].

The ribozymes of the present invention are also useful as diagnostictools to specifically or non-specifically detect the presence of atarget RNA in a sample. That is, the target RNA, if present in thesample, will be specifically cleaved by the ribozyme, and thus can bereadily and specifically detected as smaller RNA species. The presenceof such smaller RNA species is indicative of the presence of the targetRNA in the sample.

The following MATERIALS AND METHODS were used in the examples thatfollow.

Materials and Methods

Cell lines and cell culture: The 32D murine hematopoietic cell line (40)and its derivatives were grown in RPMI (Cellgro) supplemented with 12%fetal calf serum (Biofluids) and interleukin-3 (IL-3) supplied as 6%conditioned medium from the WEHI-3B murine mylomonocytic leukemia cellline.

Plasmid construction: Two synthetic single-stranded ribozymeoligonucleotides were subcloned into the mammalian vector pCR3. Thesequence and orientation of the inserts were confirmed bydideoxynucleotide sequencing of the construct using the Sequenase kit,version 2.0 (U.S. Biochemical Corp., Cleveland, Ohio). ErbB-4 ribozymesequences:

Rz 6: 5′AAU UCG GCU CAC CCA CUG AUG AGU CCG UGA GGA CGA AAC CCA AAGUCCC3′; SEQ ID NO:4

Rz 21: 5′AAU UCG UUG CCC AUC UGA UGA GUC CGU GAG GAC GAA ACA ACC UCACC3′; SEQ ID NO:5

Rz 29: 5′AAU UCC ACU AAC ACG CUG AUG AGU CCG UGA GGA CGA AAG CCU GUGACUC3′; SEQ ID NO:6

Ribozyme mediated mRNA cleavage in vitro: The substrate ErbB-4 cDNAfragment was derived by RT-PCR with RNA from MDA-MB-453 cells, whichexpress relatively high levels of ErbB-4. The PCR primers for subcloningof ErbB-4 cDNA: 5′ primer sequence: ^(5′)AAT TGT CAG CAC GGG ATC TGAGAC^(3′) (SEQ ID NO:7), and 3′ primer sequence ^(5′)GTT TCC TTA AAC AAGACC AGA TGT^(3′) (SEQ ID NO:8). The RT-PCR products were then clonedinto the PCR3 vector. Clones were sequenced to verify that theycontained the ErbB-4 cDNA fragment. We then performed in vitro run-offtranscripts from an ErbB-4 cDNA construct to generate the ErbB-4ribozyme substrate. Likewise, ribozymes were chemically synthesized asDNA oligonucleotide and subsequently synthesized in vitro by utilizingthe T7 RNA polymerase. Cleavage reactions were performed in 50 mmol/LTris-HCl, pH8.0, and 20 mmol/L MgCl₂. Substrate and ribozyme transcriptswere then mixed and incubated at 50° C. for 30 min. Reaction productswere analyzed on 6% urea polyacrylamide gel, and products were detectedby autoradiography.

Transfection by electroporation: 1×10⁷ 32D derivative cells were usedfor each transfection. 10 ug of plasmid DNA was added to cellsresuspended in 300 ul of PBS. Cells were electroporated at 250 volts,using a BioRad electroporation system, plated onto 100 mM dishes, andincubated for 24 hr. The cells were then selected in growth mediumcontaining 750 ug/ml geneticin (G418-sulfate, Gibco).

Northern blot analysis: Messenger RNA (mRNA) isolation using RNasolB(Tel-Test, Inc. Texas ). 20 ug of total RNA from each cell line wasused to hybridize with an ErbB-4 cDNA probe and autoradiographed for 48hr.

Autophosphorylation of erbB-family receptors: A total of 2×10⁸ 32Dderivative cells were washed in phosphate-buffered saline (PBS) andresuspended in 50 ml of RPMI supplemented with IL-3, and incubated for 4hr. at 37° C. Following incubation, cells were washed in PBS, andresuspended in 1 ml of PBS with Na₃(VO)₄. Remaining steps were performedon ice. Recombinant heregulin β3 isoform (EGF-like domain) was added ata final concentration of 150 ng/ml. Following a 10 min incubation, cellswere lysed in “Hepes-Lysis buffer” and the cell debris was pelleted bycentrifugation.

The lysates were then immunoprecipitated with either anti-EGFR (Ab-1,Oncogene Science, Uniondale, N.Y.), anti-erbB-2 (Ab-3, Oncogene Science,Uniondale, N.Y.), anti-erbB-3 (C17, Santa Cruz Biotechnology, SantaCruz, Calif.) or anti-erbB-4 (C18, Santa Cruz Biotechnology, Santa Cruz,Calif.) in combination with protein-A agarose (Pharmacia, Piscataway,N.J.) overnight at 4° C. with gentle agitation. Detail see elsewhere[Tang, C. K. et al. (1996) Cancer Research 56:3350-3358].

Fluorescence-activated cell sorter (FACStar) analysis: 1×10⁶ cells wereharvested and then stained for one hour with an anti-ErbB-4 monoclonalantibody (Ab-1, NeoMarker), then a secondary FITC-anti-mouse antibodywas used and the ErbB-4 level in each cell was quantitatively measuredby flow-cytometry.

Anchorage-independent growth assay: A bottom layer of 0.1 ml IMEMcontaining 0.6% agar and 10% FCS was prepared in 35 mm tissue culturedishes. After the bottom layer solidified, cells (10,000 per dish) wereadded in a 0.8 ml top layer 0.4% Bacto Agar, and 5% FCS. All sampleswere prepared in triplicate. Cells were incubated for approximately 12days at 37° C. Colonies larger than 60 um were counted in a cell colonycounter (Ommias 3600, Imaging Products Int., Inc. Charley, Va.)

Mitogenic assay: 32D transfected cells were plated at a density of 1×10⁴cells with or without IL3 supplement, or supplemented with 100 ng/ml ofHRG in the absence of IL-3. Two days post plating, the cells werelabeled with ³[H]thymidine for two hours. ³[H]thymidine incorporationwas then analyzed by β-scintillation counter.

In vitro Kinase Assay: 32D/E4, 32D/E4+V and 32D/E4+Rz29 cells were serumstarved for 2 hours prior to treatment with or without 100 ug/ml of HRG.Cells then lysed in lysis buffer. 400 ug of total protein of each cellline was used to immunoprecipitate with anti-ErbB-4 anti-body (C18,Santa Cruz Biotechnology, Santa Cruz, Calif.) in combination withprotein-A agarose (Pharmacia, Piscataway, N.J.). Reactions were carriedas described previously [Goldstein, D. J. et al. (1992) EMBO J.11:4951-4959]. Briefly, to the washed beads 50 ul of a solutioncontaining 10 mM Tris-HCL, pH 7.5, 10 mM MgCl₂, 10 mM MnCl₂, 10μCi[γ-³²P]ATP and lug aprotinin was added for 25 min at roomtemperature. Reactions were terminated by spinning down the Sepharosebeads in a microcentrifuge, discarding the supernatant and resuspendingthe beads in 50 ul SDS gel loading buffer. Eluted proteins were analyzedby SDS-PAGE and autoradiography.

In vivo studies. Athymic nude mice were inoculated subcutaneously witheither wild type cells or ErbB-4 ribozyme transfected cells. We injected5×10⁶ cells/site, two sites per mice in the presence of estrogenpellets. Because T47D cells are estrogen dependent cell line, estrogenis required for tumor growth. Estrogen pellets (60 days release) wereimplanted subcutaneously into the cervical scapular space. The size ofthe tumors were measured biweekly.

EXAMPLE 1 Generation and Demonstration of ErbB-4 Ribozyme Efficacy andSpecificity in a Cell Free System

To investigate the biological significance of ErbB-4 in human breastcancer cells, we used molecular targeting of the ErbB-4 mRNA byribozymes. Three ribozymes (Rz6, Rz21, Rz29) targeted to specific siteswithin the ErbB-4 mRNA open reading frame were generated. Theseribozymes were modeled on the previously described hammerhead structure[Zuker, M. and Stiegler, P. (1981) Nucleic Acids Res. 9:133-148; McCall,M. J. et al. (1992) Proc. Natl. Acad. Sci. USA 89:5710-5714], derivedand minimized to the catalytic center portion of 22 nucleotides. Thetargeted cleavage sites selected for the design of the ribozymes were60(Rz6), 210(Rz21) and 290(Rz29) nucleotides downstream of thetranslation initiation site of the ErbB-4 mRNA (FIG. 1). The catalyticactivity of these ribozymes was first evaluated in an extracellularsystem. All three ErbB-4 ribozymes cleaved ErbB-4 mRNA precisely andefficiently under physiological conditions in this cell free system(FIG. 2A, Lanes 2-5). Cleavage was specific as the actual sizes of thecleaved fragments correspond to the expected sizes if cleavage were tooccur immediately 3′ to the GUN sequence. As a control for specificity,catalytically inactive mutant ribozymes were engineered. Point mutationof G to A in the catalytic domain of either Rz29 or Rz6 (FIG. 2A, lanes6 and 7) resulted in loss of catalytic activity as predicted bypreviously reported mutational studies of McCall et al. (1992, supra).The specificity of these three ErbB-4 ribozymes was evaluated on anon-specific mRNA substrate. As expected, no cleavage was observedfollowing incubation of these ribozymes with ErbB-3 mRNA (FIG. 2B).These results indicate that all three of the GUN sequences chosen in theErbB-4 mRNA are accessible to ribozyme-mediated cleavage in anextracellular system.

EXAMPLE 2 An Intracellular Model System for Evaluating the Specificityand Efficacy of ErbB-4 Ribozymes

We next investigated the catalytic activity of these ribozymes in amodel cellular system. Although the ribozyme sensitivity in anextracellular system can be correlated with the predicted secondarystructure of the target RNA, the intracellular susceptibility of thetarget RNAs to ribozymes does not necessarily correlate with theirpredicted secondary structure. In addition, the complexity ofheterodimerization and transphosphorylation between the ErbB familymembers in breast cancer cells makes it difficult to determine thespecificity of ErbB-4 ribozymes. Furthermore, the goal of theseribozymes is to interrupt gene expression. If ErbB-4 is critical forcell proliferation, its down-regulation may be lethal. Thus, an idealsystem for screening the intracellular enzymatic activity of theseribozymes requires the following criteria: 1) Expression of high levelsof ErbB-4 receptor; 2) No expression of other EGF family receptors; 3)Non-lethality of ErbB-4 ribozyme introduction; and 4) Easy detection ofribozyme activity by bio-assay. We therefore used the 32D cell system toexamine the intracellular efficacy and specificity of the ErbB-4ribozymes. 32D cells are a murine hematopoietic IL3-dependent cell linethat does not express detectable levels of endogenous EGF-familyreceptors. Studies have shown that IL-3-dependence can be abrogated byintroduction of foreign growth factor receptor genes followed bystimulation with the appropriate growth factor [Pierce, J. H. (1990)Adv. Regul. Cell Growth 2:275-297]. The ability of ErbB-4-expressingcells to bypass the IL-3-dependent pathway following HRG activation[Alimandi, M. et al. (1997) EMBO J. 16: 5608-5617], provides a simplegrowth assay to determine the biological function of these ribozymesintracellularly.

EXAMPLE 3 Biological Function of EGF Family Receptors in 32D Cells

32D cell transfectants that express the EGF receptor family membersindividually and in pairwise combinations (Alimandi et al., 1997,supra). The resultant stably transfected cells were designated as32D/E1, 32D/E2, 32D/E3, 32D/E2+E3 and 32D/E4, where E1, E2, E3 and E4refer to EGFR, ErbB-2, ErbB-3 and ErbB-4 receptors, respectively. Thehigh levels of receptor expression was confirmed by Western blotting orimmunoprecipitation followed by Western blotting (data not shown). Nodetectable levels of endogenous EGF family receptor expression werefound in parental 32D cells. In the absence of cognate ligands, all ofthe 32D transfected cells remained dependent on IL-3 for survival [DiFiore, P. O. et al. (1990) Science 248:79-83]. 32D transfectants weretested for induction of IL-3-independent survival or proliferation.Consistent with previous studies (Alimandi et al., 1997, supra and DiFiore et al., 1990, supra), untransfected parental cells did notproliferate or survive following HRG stimulation. Cells transfected withErbB-4 or co-expressing ErbB-2 and ErbB-3, bypassed the IL3-dependentpathway in response to HRG stimulation, but cells transfected withErbB-2 or ErbB-3 alone did not survive and proliferated in anIL-3-dependent manner (FIG. 3). Regulation of tyrosine phosphorylationof each receptor by HRG was evaluated by immunoprecipitating thecorresponding receptors and immunoblotting with antiphosphotyrosine.FIG. 4 demonstrates that no autophosphorylation was observed in theparental cells (32D) in the presence of HRG. In both EGFR- andErbB-4-expressing cells, the receptors were constitutivelyphosphorylated; however, phosphorylation could be further inducedfollowing exposure to its cognate ligands. 32D/E2 cells demonstratedsignificant phosphorylation of ErbB-2 in the absence of HRG, butreceptor phosphorylation was not elevated in the presence of HRG (FIG.4). No phosphorylation was observed in the presence or absence of HRG in32D/E3 cells. In 32D/E2+E3 cells, a high basal level of phosphorylatedErbB-3 was observed, and increased phosphorylation was observedfollowing HRG stimulation (FIG. 4). Thus, the 32D cells provide an idealsystem to study the specificity and efficacy of the ribozymes targetingthe ErbB-family receptors.

EXAMPLE 4 Demonstration of ErbB-4 Ribozyme Catalytic Activity in 32DCells

ErbB-4 ribozymes abolish HRG-induced IL3-independence

All three ErbB-4 ribozymes were cloned into a mammalian expressionvector downstream of the CMV early promoter. We then transfected theErbB-4 Rz into 32D/E4 cells. We hypothesized that the functionalribozymes would down-regulate ErbB-4 expression and thereby reduce orabolish the HRG-induced, IL-3-independent survival or proliferation.ErbB-4 Rz transfected cells were tested for growth in the presence andabsence of HRG. Cell lines expressing one of the ErbB-4 ribozymes(Rz29), failed to respond to HRG and proliferated in an IL-3-dependentmanner. In contrast, parental 32D/E4 and vector alone-transfected cellsresponded to HRG and proliferated in the absence of IL-3. Rz6 partiallyinhibited the HRG effect. In contrast, Rz21 had no effect onresponsiveness to HRG stimulation. Table 2 summarizes the ribozymeeffects in these ErbB-4 cells. We next evaluated the specificity of theErbB-4 ribozymes by expressing all three ErbB-4 ribozymes in 32D/E2+E3cells. No effect on the HRG-induced IL3-independent survival andproliferation was observed. We then evaluated the efficacy of theribozyme by using an ErbB-2 ribozyme, which has been shown todown-regulate ErbB-2 mRNA specifically in a previous study (Personalcommunication), to target ErbB-4 mRNA. In contrast to the ErbB-4ribozyme, this ErbB-2 ribozyme did not alter the HRG-inducedIL3-independence of ErbB-4-expressing 32D cells. These data suggest thatRz6 and Rz29 are functional ribozymes, and that the effects of theseErbB-4 ribozymes are highly specific to the ErbB-4 receptor mRNA. Rz29exhibits a higher level of biological activity compared to Rz6. Rz21apparently is a non-functional ribozyme in 32D cells. The inability ofRz21 to mediate the down regulation of ErbB-4 may be due to severalpossibilities. For example, the target site may not accessableintracellularly, or Rz21 may be unstable in 32D cells.

TABLE 2 Effect ot ErbB-4 ribozymes on the density of 32D/E4 cells inresponse to IL-3 starvation and HRG stimulations Number of viable cells(×1000 cells/ml) Cell line −IL-3 +IL-3 +HRG/−IL-3 E4 1.3 1996 1490E4/Vector 1 1894 1369 E4/Rz6 1.1 1717  367 E4/Rz21 1 1845 1300 E4/Rz291.2 1823  56 E4/ErbB-2 ribozyme 1.2 1798 1279 E2 + E3/Rz6 1.1 1869 1307E2 + E3/Rz21 1 1946 1377 E2 + E3/Rz29 1.2 1854 1298

EXAMPLE 5 ErbB-4 Ribozyme Abolishes the HRG Stimulation of Mitogenesis

To confirm the growth inhibitory activity of the ErbB-4 ribozymes, amitogenic assay to measure DNA synthesis was performed on ErbB-4Rz-transfected cells. As shown in FIG. 5, all the 32D transfected cellsexhibited very low levels of [³H]thymidine incorporation in the absenceof IL-3. In contrast, all the 32D transfected cells exhibited highlevels of [³H]thymidine incorporation in the presence of IL-3, asexpected. In the 32D/E4 control cells, HRG stimulated high levels of[³H]thymidine incorporation in the absence of IL-3; whereas the[³H]thymidine incorporation was almost completely abolished in the Rz29-transfected cells. [³H]thymidine incorporation was significantly reducedin Rz6-transfected cells, but to a lesser extent than inRz29-transfected cells. No significant changes in the Rz21-transfectedcells were observed. These results were thus consistent with the growthassay.

EXAMPLE 6 ErbB-4-Rz-mediated Down-regulation of ErbB-4 Expression in32D/ErbB-4 Cells

To evaluate the intracellular enzymatic cleavage activity of ErbB-4ribozymes, the ribozyme transfectants were examined for ErbB-4 mRNAlevels by Northern blot analysis. Rz6- and Rz29-expressing cellsexhibited significantly reduced ErbB-4 mRNA levels relative to controlcells or to Rz21-expressing cells (data not shown). Thus, theabolishment of the HRG-induced IL-3 independent biological effectcorrelates with reduction of ErbB-4 mRNA levels in these cells.

To further characterize the ribozyme effect, we quantitatively examinedthe ErbB-4 ribozyme-mediated down-regulation of ErbB-4 receptorexpression in these ErbB-4Rz transfected cells by FACS analysis.Consistent with Northern analysis, Rz29- and Rz6-transfected cellsexpressed significantly less cell surface ErbB-4 receptor relative tothe 32D/E4 control cells (65% and 45% less ErbB-4, respectively; FIG.6). No significant reduction of ErbB-4 expression was detected inR21-transfected cells. Taken together, these data suggest that theErbB-4 Rz29 and Rz6 are biologically functional ribozymes.

EXAMPLE 7 Reduction of Autophosphorylation by ErbB-4 Ribozymes

To determine whether the HRG-induced IL-3-independent phenotype inErbB-4 transfectants correlated with an increase in receptor tyrosinephosphorylation, the autophosphorylation of the receptors in these cellswas examined by a kinase assay. FIG. 7 demonstrates that the level ofErbB-4 intrinsic tyrosine kinase activity in Rz29-transfected cells wasmarkedly reduced compared to control transfectants (32D/E4 and32D/E4/Vector). Because ErbB-4 expression was down-regulated only 65% byRz29, the cells still express ErbB-4 receptors. HRG was therefore stillable to induce the phosphorylation of the remaining ErbB-4 receptors.However, the level of phosphorylation was significantly lower than the32D/E4 cells or the vector transfected cells (32D/E4/V). Reduction ofphosphorylation correlated with reduction in expression of ErbB-4.Furthermore, these data also imply that while Rz29 is specificallycleaving its target mRNA, it does not affect the function of thosereceptors that are expressed. Taken together, these intracellularexperiments demonstrated that the decrease of ErbB-4 protein production,activation and mRNA expression correlate with the ErbB-4 ribozymecatalytic activity.

EXAMPLE 8 The Effect of Down-regulation of ErbB-4 Receptor in HumanBreast Cancer Cells

To investigate the biological and biochemical functions of ErbB-4 inhuman breast cancer, we expressed the ErbB-4 ribozymes in severalErbB-4-positive human breast cancer cell lines. Four human breast cancercell lines were selected as recipient cells: T47D, MCF-7, MDA-MB-453 andMDA-MB-231. In T47D and MCF-7 cells, there is a relatively high level ofErbB-4 receptor expression and a moderate level of other EGF-familyreceptors, whereas MDA-MB-453 cells express low endogenous levels ofErbB-4 and high levels of ErbB-2 and ErbB-3. MDA-MB-231 expresses a highlevel of EGFR and a relatively low level of ErbB-2, but does not expressdetectable level of ErbB-3 or ErbB-4. The functional ErbB-4 ribozymes,as well as a control vector, were introduced into these cell lines bystable transfection. The sublines T47D/Rz, MCF-7/Rz, MDA-MB-453/Rz andMDA-MB-231/Rz as well as empty vector control cell lines wereestablished. We then assessed the ribozyme mediated down-regulation ofErbB-4 expression by FACS analysis. FIG. 9 illustrates that ErbB-4ribozyme capable of down-regulation of endogenous ErbB-4 expression by50% and 70% in two of the ribozyme transfected T47D pooled populationclones, T47D/Rz-poolA and T47D/Rz-pool 20, respectively. We also foundthat the ErbB-4 expression was almost completely down-regulated in someof the ErbB-4 ribozyme transfected MCF-7 cells, such as MCF-7/RzA4 andMCF-7/RzB1 clones (data not shown), as well as in ribozyme transfectedMDA-MB-453 cells (data not shown). However, no effect was observed onother EGF family receptors in these ErbB-4 ribozyme transfected cells,respectively (Data not shown). Furthermore, ribozyme-mediateddown-regulation of ErbB-4 receptor expression was confirmed by reductionof ErbB-4 mRNA by Northern blot analysis (data not shown).

EXAMPLE 9 Reduction of NRG and BTC Induced ErbB-4 Autophosphorylation inT47D/Rz Transfected Cells

We next determined whether NRG or BTC-induced ErbB-4 receptor tyrosinephosphorylation was affected by reduction of ErbB-4 expression inribozyme transfected cells. Phosphorylation experiments were performedon ribozyme transfected clones. FIG. 10 demonstrates that the level ofErbB-4 intrinsic tyrosine kinase activity in T47D/Rz6 Pool 20transfected cells was markedly reduced when compared with controltransfectants (T47D/wt and T47D/Vector) cells. Reduction ofphosphorylation correlates with a reduction in ErbB-4 expression level.A similar effect was observed in BTC-induced ErbB-4 tyrosinephosphorylation. These experiments demonstrate that the reduction ofErbB-4 activation correlates with down-regulation of ErbB-4 proteinproduction.

EXAMPLE 10 Down-regulation of ErbB-4 in Cell Lines Expressing RelativelyHigh Level of ErbB-4 Resulted in an Inhibition of Colony Formation

In order to assess the biological significance of ErbB-4 in human breastcancer, we evaluated the in vitro growth of ErbB-4 ribozyme transfectedT47D, MCF-7, MDA-MB-453 and MDA-MB-231 cells by anchorage-dependent aswell as anchorage-independent growth assays. Down-regulation of ErbB-4expression in cell lines expressing a relatively high level of ErbB-4(T47D and MCF-7 cells) resulted in an inhibition of colony formationthat was independent of colony size. FIG. 11 illustrates thatdown-regulation of ErbB-4 by 50% in T47D/Rz-poolA cells displayed a 50%reduction in their ability to form colonies in soft agar. Colonyformation was almost completely abolished in T47D/Rz-pool20 cells, whichhad an 80% down-regulation of ErbB-4, indicating a partial reversion oftransformation. Furthermore, inhibition of colony formation wasindependent of threshold colony size. A similar phenotype was observedin ribozyme transfected MCF-7 cells (FIG. 12). These data demonstratedthat inhibition of growth is correlated with the level ofdown-regulation of ErbB-4 in these ribozymes transfected cells. However,growth inhibition was not observed in MDA-MB-453/Rz cells, which expresslow levels of ErbB-4 and high levels of ErbB-2 and ErbB-3.Interestingly, FACS analysis revealed that the expression of the ErbB-4receptor was completely abrogated by the ErbB-4 ribozyme in these cellsas well (Data not shown). In a parallel experiment, we verified thespecificity and efficacy of the anti-ErbB-4 ribozymes with MDA-MB-231cells, which do not express detectable level of ErbB-4. Obviously, noeffect was observed in ribozyme transfected MDA-MB-231 cells,respectively (Data not shown). These data suggest that the biologicaleffect of ErbB-4 receptor expression is dependent upon its relativelevels in a given cell line.

EXAMPLE 11 The Sensitivity of Biological Responses to Different EGF-likeLigands is Dependent upon the Relative Level of ErbB Family Receptors

Regulation of ErbB receptor family members activation is very complex. Alarge number of ErbB ligands have been described (reviewed in Peles andYarden (1993) BioEssays 15: 815-824; Groenen et al., (1994) GrowthFactors 11:235-257; Salomon et al., (1995) Crit. Rev. Oncol.-Hematol.19: 183-232; Pinkas-Kramarshi et al. (1997) J. Mammary Gland Biol.Neoplasia 2:97-107]. We next compared the effects of EGF-like ligandsbetween ribozyme transfected T47D cells (T47D/Rz) and T47D/wt. Weobserved that neuregulin induced colony formation was significantlyinhibited in T47D/Rz transfected cells. Down-regulation of ErbB-4 inT47D cells reduced NRG stimulated colony formation by 80%. In contrast,wild type T47D cells exhibited an 11-fold increases in colony formationwhen treated with neuregulin appears to have the most dominant effectamong the six of EGF-like ligands. Betacellulin, which predominantlybinds to EGFR and can also activate the ErbB-4 and ErbB-2/ErbB-3heterodimers, had the most dominant effect on the induction of colonyformation, when compared with the other EGF-like ligands (FIG. 13).These data demonstrate that NRG was significantly more active than otherEGF-like ligands in T47D wild type cells, while down-regulation ofErbB-4 in T47D cells revealed almost complete abrogation of the NRGactivity, suggesting that NRG signaling occurs primarily through ErbB-4in T47D cells. Interestingly, BTC was comparable to NRG stimulatingcolony formation by nearly six fold in T47D wild type cells and is thedominant ligand in ErbB-4 depleted T47D cells. These results suggestedthat altering the expression of ErbB-family receptors in the cellresults in an alteration in the biological activities of EGF-relatedpeptides.

EXAMPLE 12 Inhibition of Tumor Formation in Nude Mice

Down-regulation of ErbB-4 led to dramatic effects on anchorage-dependentand anchorage-independent growth in MCF-7 and T47D cells. We nextexplored the in vivo effects of down-regulation of ErbB-4 in MCF-7 andT47D cells. MCF-7 or T47D wild type cells (5×10⁶) as well as theribozyme transfected cells were implanted in ovariectomized mice. Withestradiol treatments, the T47D wild type cells grew to a mean tumor sizeof 500±20 mm³ (FIG. 14; filled circles). In contrast, tumor growth ofribozyme expressing T47D cells was significantly inhibited (p<0.001;student's t test) with a mean tumor size of 80±14 mm³ (FIG. 14;triangles and squares). Moreover, tumor growth of T47D cells transfectedwith the catalytically inactive ribozyme (Rz21) was not significantlydifferent from control cells (data not shown). Similar experiments wereperformed with ribozyme transfected MCF-7 cells. FIG. 15 demonstratedthat down-regulation of ErbB-4 expression in MCF-7 cells dramaticallyreduced the tumor formation. With estradiol treatments, the MCF-7 wildtype (MCF-7/wt) and an empty vector transfected MCF-7 cells(MCF-7/vector) grew large tumors with a mean tumor size of 2400±270 mm³.In contrast, tumor growth of ribozyme expressing MCF-7 cells wasdrastically inhibited (p<0.0003; student's t test) with a mean tumorsize of 580±74 mm³ (p<0.001; student's t test). Table 3 summarizes thein vitro and in vivo effects of down-regulation of ErbB-4 in humanbreast cancer cell lines.

TABLE 3 Selective growth inhibition with ribozyme- mediateddown-regulation of ErbB-4 in breast cancer cells^(a) Expression levelsof Effects of down-regulation EGF-family receptors of ErbB-4 Cell %inhibition of % inhibition of line EGFR ErbB-2 ErbB-3 ErbB-4 ER colonyformation tumorigenicity MCF-7 + ++ +++ ++++ + 60-80 70 T47D ++ ++ +++++++ + 50-70 50-60 453 +/− ++++ +++ − − 0 N/A 231 ++++ + +/− − − 0 N/A453 = MDA-MB-453 231 = MDA-MB-231 The expression levels of ErbB-familyreceptors were determined by FACS analysis.

EXAMPLE 13 Expression of ErbB-4 in Primary Breast Carcinomas

We next investigated the frequency of ErbB-4 expression in breastcarcinomas using immunohistochemical analysis with an anti-ErbB-4monoclonal antibody. The expression of ErbB-4 was analyzed in 50 primarybreast carcinomas. The results showed expression of ErbB-4 in 70% of thetotal samples (35 of 50) examined. Interestingly, 80%(28 of 35) of theErbB-4 positive samples were estrogen receptor positive (ER+) breastcarcinomas and 67% (10 of 15) of the negative or weak ErbB-4 expressionswere estrogen receptor negative (ER−) breast carcinomas (Table 4). Itappears that there is a statistically significant (P=0.001) directcorrelation between the expression of estrogen receptors and theexpression of ErbB-4. We also surveyed the ErbB-4 expression in humanbreast cancer cell lines by FACS analysis. Surprisingly, most of ER+cell lines expressed relatively high levels of ErbB-4 and ER− cell linesexpressed low levels or non-detectable levels of ErbB-4

TABLE 4 Correlation of ErbB-4 expression with prognostic factors inbreast cancer ER PR ErbB-4 expression − + − + weak/negative (+/−) 10  59 6 positive (++/+++)  7 28 9 26  PR = Progesterone receptor n = 50

DISCUSSION

In this study, we generated three specific hammerhead ribozymes (Rz)targeted to ErbB-4 mRNA. We have demonstrated that these ErbB-4ribozymes (Rz6, Rz21, Rz29) effectively catalyze precise cleavage ofErbB-4 mRNA under physiological conditions in an extracellular system(FIG. 2). Furthermore, we demonstrated that these ribozymes do notcleave mRNA of other EGFR family members, despite the high degree ofsequence homology shared by these receptors. Point mutation of theseErbB-4 ribozymes in the catalytic domain resulted in loss of catalyticactivity and failure to cleave ErbB-4 mRNA. These inactive ribozymeshave identical binding arms to the active version but have a mutatedcatalytic domain. Thus, these mutated versions are capable of binding tothe target sequence but are not able to cleave the target mRNA. Takentogether, these control experiments demonstrate that the ErbB-4ribozymes are highly specific for the ErbB-4 mRNA.

Using the 32D cell system to study the intracellular enzymatic activityof ErbB-4 ribozymes, we clearly demonstrated that the ribozymes arespecific and effectively downregulate the EGF receptor family members.In this system, one ErbB-4 ribozyme (Rz29) significantly reduced theErbB-4 mRNA level and down-regulated ErbB-4 receptor expression (FIG.6), thereby reversing the HRG-induced IL3-independent phenotype of32D/E4 cells (table 2). Rz6 partially down-regulated the expression ofthe ErbB-4 receptor, and somewhat blocked the IL3-independent phenotype.In contrast, Rz21 failed to down-regulate the ErbB-4 expression andinhibit the mitogenic response to HRG treatment in 32D/ErbB-4 cells. Itis clear from these data that not all of the sites tested are equallyamenable to intracellular ribozyme-mediated cleavage. This is in spiteof the fact that ribozymes to all of the sites were shown to becatalytically active extracellular biochemical assays. RNA secondarystructure or association with cellular proteins may affect target siteaccessibility. We therefore investigated the specificity and efficacy ofthese ribozymes in a well-defined cellular system. Two sets ofexperiments were conducted to control for ribozyme specificity andefficacy intracellularly. Due to the high homology between the EGFreceptor family members, the intracellular specificity of ErbB-4ribozymes was demonstrated using the 32D cells ectopically co-expressingErbB-2 and ErbB-3. None of the ErbB-4 ribozymes (Rz6, Rz21, Rz29) hadany effect on the level of ErbB-2 or ErbB-3 expression or theHRG-induced IL-3-independent phenotype in these 32D derivative cells(Table 2). Moreover, an ErbB-2 ribozyme, previously shown todown-regulate the expression of ErbB-2 mRNA, failed to decrease ErbB-4expression in 32D/ErbB-4 cells. The lack of down-regulation of ErbB-4expression in these control experiments is evidence of the high degreeof specificity of these ribozymes. Furthermore, in the absence of HRG,cells expressing these ribozymes remained strictly dependent on IL3 forgrowth. In contrast, two ErbB-4 ribozymes (Rz29 and Rz6) decreased theHRG-induced, IL-3-independent proliferation. Taken together, thesephenomena indicate that only the ErbB-4 transcript is directly affectedby these ribozymes. Although the ErbB-4 expression was reduced in Rz6-and Rz29-transfected 32D/E4 cells, the remaining ErbB-4 receptors inthese cells were still phosphorylated in response to HRG treatment (FIG.7). This characteristic provides strong support for a cleavage-mediatedmechanism of action for the ribozymes. Therefore, the constructed ErbB-4Rz29 and Rz6 are biologically functional ribozymes and are highlyspecific for the targeted ErbB-4 mRNA in 32D cells.

To evaluate the effects of down-regulation of ErbB-4 in anErbB-4-positive human breast cancer line, Rz29 was transfected into T47Dcells. Down-regulation of ErbB-4 receptor in T47D cells resulted inreduction of colony formation in anchorage-independent assay and intransfection efficiency compared to vector- or Rz21-transfected cells.The low efficiency of Rz6 and Rz29-expressing, drug-selected clones isunlikely due to a non-specific effects, since all the ribozymes werecloned into the same vector. Furthermore, Rz6 and Rz29 onlydown-regulated ErbB-4 but not other ErbB-receptor family members.Reduction of colony formation suggests that ErbB-4 expression andmitogenic signaling may be essential for T47D cell survival. Currently,we are conducting these studies using an inducible promoter system.These preliminary findings suggest that down regulation of ErbB-4expression, as shown by FACS, diminished the ErbB-4-mediatedintracellular signaling. Because of heterodimerization between thefamily receptors, down-regulation of ErbB-4 receptor may also indirectlyinterrupt receptor signaling pathways initiated by other family members.This could result in diminished tumorigenicity in T47D cells. Theseresults also show that our ribozyme is active in a human carcinoma cellline.

32D cells are strictly dependent upon interleukin-3 (IL-3) for survivaland proliferation. However, HRG was capable of stimulating its cognatereceptors, coupling to cellular signaling pathways in 32D derivativesand thereby abrogating the IL-3 dependence of these cells. Using theErbB-4 ribozymes in 32D cell system, we provide the first evidence thatthe different threshold levels of ErbB-4 expression and activationcorrelate with different responses to HRG stimulation. High levels ofErbB-4 expression, phosphorylation and homodimerization are necessaryfor HRG stimulated IL3-independent cell proliferation in the 32D/E4cells. Low levels of ErbB-4 expression allows HRG-inducedphosphorylation, but are insufficient to couple the receptor activationto cellular signaling, particularly in the case of Rz29-transfected32D/E4 cells. In line with these observations, in a recent study usingBa/F3 cell derivatives, HRG failed to induce the IL-3-independentpathway in the ErbB-4 transfected cells [Riesell, D. J. et al. (1996)Oncogene 12:345-353]. It is possible that the level of ErbB-4 expressionin these Ba/F3/ErbB-4 cells is lower than our 32D/E4 cell line. Wedemonstrate that the IL-3-independent pathway appears to be verysensitive to the amount of ErbB-4 expression, as well as the tyrosinephosphorylation level. The Rz6-transfected cells, whose ErbB-4expression was down-regulated by 45%, exhibited a weak response to HRG,whereas the Rz29-transfected cells, whose ErbB-4 expression level wasdown-regulated by 65%, failed to respond HRG stimulation. HRG was stillable to induce ErbB-4 receptor phosphorylation in these cells, but thelevel of phosphorylation was much lower than in the 32/E4 cells. Thislevel of phosphorylation is not sufficient to stimulate the cellularresponse. These results also suggest that homodimers of ErbB-4 cantransmit biological signals. This is consistent with a previous reportthat ErbB-4 homodimers constitute a functional HRG receptor [Plowman, G.D. et al. (1993) Nature 366:473-475]. HRG can induce 32D/ErbB-2+ErbB-3cells to bypass the IL3-dependent pathway, presumably due totransphosphorylation and cross talk between the receptors throughheterodimerization of ErbB-2 and ErbB-3. These results are consistentwith previous studies concerning ErbB receptor transphosphorylation(40). While ErbB-3 appears to be a defective tyrosine kinase receptor,it mediates HRG signals through heterodimer formation with either EGFRor ErbB-2 (Plowman et al., 1993, supra). Furthermore, almost all of thebreast cancer cell lines express more than one of the EGFR familymembers. These results imply that inter-receptor cross-talk may play animportant role in human breast cancer.

In this study, we employed ribozyme technology to achieve the functionalgene “knockout” strategy to define the role and biological significanceof ErbB-4 in human breast cancer. We demonstrated that the ErbB-4ribozyme is capable of down-regulation of endogenous ErbB-4 expressionin several human breast cancer cell lines, but no effect was observed onother members of the EGF receptors family. In stably mass-transfectedT47D cells, ErbB-4 ribozyme expression depleted ErbB-4 mRNA and proteinlevels by 50-75%. This inhibition is even more remarkable whenconsidering that mass-transfected cells (and not clonal subpopulations)were used. This substantial inhibition enabled us to begin a novel studyof the effects of a functional ErbB-4 knockout on in vitro and in vivotumor growth of breast cancer cells. We observed that down-regulation ofErbB-4 in T47D and MCF-7 cells which express relatively high levels ofErbB-4 significantly inhibited colony formation. In addition,down-regulation of ErbB-4 in T47D cells significantly impairedNRG-induced ErbB-4 phosphorylation. However, complete depletion ofErbB-4 did not affect the anchorage-dependent and anchorage-independentgrowth in MDA-MB-453 cells, which express low levels of endogenous ErbB4and high levels of ErbB-2 and ErbB-3. Furthermore, down-regulation ofErbB-4 in T47D and MCF-7 cells significantly inhibited tumor formationin athymic nude mice with P<0.001, P<0.0003. These data provide thefirst evidence that elevation of ErbB-4 expression plays a proliferationrole in vitro and in vivo in some human breast cancer cell lines (T47D,MCF-7). These data suggest that inhibition of growth was observed whenover expressed receptors were targeted. Furthermore, ErbB receptorsundergo extensive heterodimerization. The inactivation or blocking ofErbB-4 signaling may also disrupt and diminish the EGFR or ErbB-2signaling pathways, through heterodimerization with ErbB-4. A similarconclusion was reported by Hynes and her colleagues, who found thatblocking cell surface expression of ErbB-2 and EGFR by intracellularexpression of a single-chain antibody specific for ErbB-2 (scFv-5R) andEGFR (scFv-R1R) led to only a slight reduction in colony formation ofT47D cells, which express low levels of ErbB-2 and EGFR. However, inMDA-MB-468 cells, scFv-5R and scFvR1R inhibited colony formation by 90%and 94%, respectively. MDA-MB-468 express high levels of EGFR and TGFα,treatment with a Mab which competes with ligand binding and inhibitscell growth, indicating that these cells are dependent upon an autocrineloop for growth. Despite the fact that these cells have very low levelsof ErbB-2, inhibition of colony formation by scFvR suggests that TGFαactivated heterodimers of EGFR and ErbB-2 provide the major growthstimulus to these cells [Jannot, C. B. et al. (1996) Oncogene18:275-282; Beerli, R. R. et al. (1995) Mol. Cell Biol. 15:6496-6505].These data also suggest that depending upon the cellular context, itseems that not only the presence or absence of a specific EGF-familyreceptor in a given cell line influence the nature of cellproliferation, but also the relative expression level of theErbB-receptors determines the roles of these receptors in a given cellline. Over expression or a relatively high level of an ErbB-receptorplays a role in breast cancer proliferation. In general, inhibition ofgrowth was observed when over expressed receptors were targeted.

Regulation of ErbB-receptor family members activation is very complex.ErbB receptors undergo extensive heterodimerization which makesligand-induced signaling even more complex. We show that NRG-stimulatedphosphorylation of ErbB-4 was significantly reduced and NRG inducedcolony formation was substantially reduced from 11 fold to only 2.5 foldin ribozyme transfected T47D cells (T47D/Rz), indicating that the majorNRG signaling was through ErbB-4. It implies that NRG signaling throughErbB-2/ErbB-3 heterodimers may play a minor role in T47D cells due totheir low expression levels. BTC, a ligand for EGFR, ErbB-4, and alsoErbB-2/ErbB-3 heterodimers, exhibited the most dominant effect oninduction of colony formation among the EGF-like ligands in T47D/Rztransfectants (FIG. 13). These data indicated that down-regulation ofErbB-4 only partially affects the BTC signaling. Although, BTC signalingthrough ErbB-4 may be blocked, BTC's may be able to elicit signallingvia other ErbB family receptors. These data suggested that altering theexpression of ErbB-family receptors in the cell results in altering thebiological activities by EGF-related peptides. EGF-related growthfactors show distinguishable biological activities, most likelydepending on the subsets of ErbB-receptors that become activated.

In addition, we have also investigated the expression of ErbB-4 inprimary breast carcinoma, using immunohistochemical analysis with ananti-ErbB-4 monoclonal antibody. ErbB-4 expression was found in 70% ofthe 50 samples examined. Overexpression of ErbB-4 is correlated with ER+and progesterone receptor positive (PgR+) primary breast tumors.Although the data are incomplete, a pattern is suggesting that ErbB-4may be a favorable prognostic factor. It is interesting thatoverexpression of ErbB-4 is correlated with ER expression, unlike otherEGF-family receptors. It will be intriguing to define the mechnism bywhich ErbB-4 expression maintains ER expression in human breast cancer.

In conclusion, our data suggest that the role and function of EGF-familyreceptors in breast cancer cells dependent on the relative levels ofexpression of the EGFR, ErbB-2, and ErbB-4 rather than the absolutelevels of any single ErbB family receptors expression.

Our studies provide strong evidence that ribozymes (Rz6, Rz29)specifically target ErbB-4 mRNA for degradation extracellularly andintracellularly. These functional ErbB-4 ribozymes should provideimportant tools for delineating the biological and biochemicalconsequences of ErbB-4 expression in human breast cancer cells.Furthermore, our study supports the potential for using ribozymes astherapeutic agents for human breast cancer (Gassmann, M et al. (1995)Nature 378: 390-394; Lieber, A. et al. (1996) J. Virol. 70:8782-8791;Grassi, G. and Marini, J. C. (1996) Ann. Med. (England) 28: 499-510;Birikh, K. R. et al. (1997) Eur. J. Biochem 245:1-16; Prislei, S. et al.(1997) RNA 3: 677-687).

8 1 19 RNA Artificial Sequence Description of Artificial Sequence mRNAtarget sequence 1 gacuuugggu cugggugag 19 2 16 RNA Artificial SequenceDescription of Artificial Sequence mRNA target sequence 2 ugagguugucaugggc 16 3 19 RNA Artificial Sequence Description of ArtificialSequence mRNA target sequence 3 gucacaggcu acguguuag 19 4 49 RNAArtificial Sequence Description of Artificial Sequence Syntheticribozyme sequence 4 aauucggcuc acccacugau gaguccguga ggacgaaacccaaaguccc 49 5 47 RNA Artificial Sequence Description of ArtificialSequence Synthetic ribozyme sequence 5 aauucguugc ccaucugaug aguccgugaggacgaaacaa ccucacc 47 6 49 RNA Artificial Sequence Description ofArtificial Sequence Synthetic ribozyme sequence 6 aauuccacua acacgcugaugaguccguga ggacgaaagc cugugacuc 49 7 24 DNA Artificial SequenceDescription of Artificial Sequence Primer 7 aattgtcagc acgggatctg agac24 8 24 DNA Artificial Sequence Description of Artificial SequencePrimer 8 gtttccttaa acaagaccag atgt 24

What is claimed is:
 1. An enzymatic RNA molecule which specificallycleaves mRNA produced from the gene ErbB-4, wherein the enzymatic RNAmolecule specifically cleaves RNA sequence consisting of any of SEQ IDNOs 1-3, and wherein said enzymatic RNA is in a hammerhead motif.
 2. Anenzymatic RNA molecule which specifically cleaves mRNA produced from thegene ErbB-4, wherein the enzymatic RNA molecule specifically cleaves RNAsequence consisting of any of SEQ ID NOs 1-3, and wherein said enzymaticRNA is in a hammerhead motif and consisting of any sequence selectedfrom the group consisting of SEQ ID NOs 4-6.